Myelin is a dynamic, functionally active membrane in bi-directional communication with the axon and receiving and processing signals from the environment. We propose that a key to understanding the active biological functions of myelin is to identify and characterize functionally the complete repertoire of myelin proteins, i.e., the Myelin Proteome. We have undertaken an extensive proteomic analysis of myelin, developed a 2D-PAGE map of myelin proteins, identified to date 125 of these proteins, and illustrated the power and utility of this approach through specific applications of comparative proteomics. This proteomic map and related conceptual approaches offers a major advance for investigations into the diverse biological roles of the myelin membrane and suggest a paradigm for similar studies in other neural systems. The long range goals of this project are to identify and characterize the molecular components of the myelin membrane;develop a model for the arrangement of these molecules in the myelin membrane, with a particular focus on the role of glycosphingolipid-cholesterol microdomains;assign functional correlates to proteins central to myelin biogenesis, maintenance and activity;and relate these concepts to a better understanding of de/remyelination in multiple sclerosis and other demyelinating diseases. Myelin Protein Identity: In Aim I we further develop the Myelin Proteome, applying new approaches for identification of unresolved proteins. This study is providing an invaluable myelin protein database for the myelin biology community at large, forming the basis for numerous projects. Myelin Protein Function: In Aim II we investigate OL differentiation- and physiology-mediated changes in protein expression using Direct In-Gel Electrophoresis (DIGE) and Isotope-Coded Affinity Tag (ICAT) mass spectrometric methodologies, and manipulation of protein expression and function using antibody blocking, RNAi technology and genetically modified mouse mutants. Myelin Protein Disease: In Aim III, we study mechanisms by which cell signaling pathways become activated by environmental factors in normal OL physiology and in myelin biology disease. This focuses on proteomic analyses of antibody perturbations that mimic natural ligands and neuroimmunological disease conditions, including the roles of Myelin Oligodendrocyte Glycoprotein (MOG), galactosphingolipids, and glycosphingolipid-cholesterol microdomains in the initiation of signal transaction.